CA2577473C - A lipase powder composition and a process for preparing an esterified compound by using the same - Google Patents
A lipase powder composition and a process for preparing an esterified compound by using the same Download PDFInfo
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- CA2577473C CA2577473C CA2577473A CA2577473A CA2577473C CA 2577473 C CA2577473 C CA 2577473C CA 2577473 A CA2577473 A CA 2577473A CA 2577473 A CA2577473 A CA 2577473A CA 2577473 C CA2577473 C CA 2577473C
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- lipase
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- 108090001060 Lipase Proteins 0.000 title claims abstract description 196
- 102000004882 Lipase Human genes 0.000 title claims abstract description 196
- 239000004367 Lipase Substances 0.000 title claims abstract description 196
- 235000019421 lipase Nutrition 0.000 title claims abstract description 195
- 239000000203 mixture Substances 0.000 title claims abstract description 24
- 150000001875 compounds Chemical class 0.000 title claims abstract description 15
- 238000004519 manufacturing process Methods 0.000 title claims description 13
- 239000000843 powder Substances 0.000 title description 20
- 241000588810 Alcaligenes sp. Species 0.000 claims abstract description 16
- 241000228168 Penicillium sp. Species 0.000 claims abstract description 12
- 241000135252 Rhizomucor sp. Species 0.000 claims abstract description 12
- 230000001476 alcoholic effect Effects 0.000 claims abstract description 7
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 7
- 241000952054 Rhizopus sp. Species 0.000 claims abstract description 6
- 241001285933 Thermomyces sp. Species 0.000 claims abstract description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 22
- 238000005886 esterification reaction Methods 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000000034 method Methods 0.000 claims description 16
- 230000032050 esterification Effects 0.000 claims description 13
- 235000011187 glycerol Nutrition 0.000 claims description 11
- 239000002245 particle Substances 0.000 claims description 11
- 241000235403 Rhizomucor miehei Species 0.000 claims description 9
- 150000001735 carboxylic acids Chemical class 0.000 claims description 9
- 241000228147 Penicillium camemberti Species 0.000 claims description 2
- 235000002245 Penicillium camembertii Nutrition 0.000 claims description 2
- 239000000969 carrier Substances 0.000 claims description 2
- 235000021122 unsaturated fatty acids Nutrition 0.000 claims description 2
- 150000004670 unsaturated fatty acids Chemical class 0.000 claims description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 abstract 1
- 239000002994 raw material Substances 0.000 description 18
- 239000007864 aqueous solution Substances 0.000 description 16
- 239000000243 solution Substances 0.000 description 15
- 102000004190 Enzymes Human genes 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 13
- 239000012528 membrane Substances 0.000 description 12
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 7
- 238000000108 ultra-filtration Methods 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000001035 drying Methods 0.000 description 6
- 230000003287 optical effect Effects 0.000 description 6
- 239000012254 powdered material Substances 0.000 description 6
- JBYXPOFIGCOSSB-GOJKSUSPSA-N 9-cis,11-trans-octadecadienoic acid Chemical compound CCCCCC\C=C\C=C/CCCCCCCC(O)=O JBYXPOFIGCOSSB-GOJKSUSPSA-N 0.000 description 5
- 229940108924 conjugated linoleic acid Drugs 0.000 description 5
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 5
- 239000007921 spray Substances 0.000 description 5
- 238000001694 spray drying Methods 0.000 description 5
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000007664 blowing Methods 0.000 description 3
- 210000005056 cell body Anatomy 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 235000014113 dietary fatty acids Nutrition 0.000 description 3
- 239000000194 fatty acid Substances 0.000 description 3
- 229930195729 fatty acid Natural products 0.000 description 3
- 150000004665 fatty acids Chemical class 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 150000005846 sugar alcohols Polymers 0.000 description 3
- 238000005809 transesterification reaction Methods 0.000 description 3
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 108010048733 Lipozyme Proteins 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 240000005384 Rhizopus oryzae Species 0.000 description 2
- 235000013752 Rhizopus oryzae Nutrition 0.000 description 2
- 241000223258 Thermomyces lanuginosus Species 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 238000002523 gelfiltration Methods 0.000 description 2
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 2
- 235000019626 lipase activity Nutrition 0.000 description 2
- FCCDDURTIIUXBY-UHFFFAOYSA-N lipoamide Chemical compound NC(=O)CCCCC1CCSS1 FCCDDURTIIUXBY-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 238000001471 micro-filtration Methods 0.000 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- -1 triglyceride Chemical class 0.000 description 2
- OYHQOLUKZRVURQ-NTGFUMLPSA-N (9Z,12Z)-9,10,12,13-tetratritiooctadeca-9,12-dienoic acid Chemical compound C(CCCCCCC\C(=C(/C\C(=C(/CCCCC)\[3H])\[3H])\[3H])\[3H])(=O)O OYHQOLUKZRVURQ-NTGFUMLPSA-N 0.000 description 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- GYSCBCSGKXNZRH-UHFFFAOYSA-N 1-benzothiophene-2-carboxamide Chemical compound C1=CC=C2SC(C(=O)N)=CC2=C1 GYSCBCSGKXNZRH-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- GHVNFZFCNZKVNT-UHFFFAOYSA-N Decanoic acid Natural products CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 description 1
- 239000005639 Lauric acid Substances 0.000 description 1
- 241001558145 Mucor sp. Species 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 235000021314 Palmitic acid Nutrition 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 150000008044 alkali metal hydroxides Chemical class 0.000 description 1
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 1
- DTOSIQBPPRVQHS-PDBXOOCHSA-N alpha-linolenic acid Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(O)=O DTOSIQBPPRVQHS-PDBXOOCHSA-N 0.000 description 1
- 235000020661 alpha-linolenic acid Nutrition 0.000 description 1
- 150000001414 amino alcohols Chemical class 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 150000007942 carboxylates Chemical class 0.000 description 1
- 239000003729 cation exchange resin Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000006356 dehydrogenation reaction Methods 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 239000012051 hydrophobic carrier Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 229960004488 linolenic acid Drugs 0.000 description 1
- KQQKGWQCNNTQJW-UHFFFAOYSA-N linolenic acid Natural products CC=CCCC=CCC=CCCCCCCCC(O)=O KQQKGWQCNNTQJW-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 235000021313 oleic acid Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 229920002239 polyacrylonitrile Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000005185 salting out Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- TUNFSRHWOTWDNC-HKGQFRNVSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCC[14C](O)=O TUNFSRHWOTWDNC-HKGQFRNVSA-N 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6454—Glycerides by esterification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
There is provided a lipase composition comprising (a) a powdered lipase which is a lipase derived from Rhizomucor sp. or a powdered lipase which is a lipase derived from Penicillium sp. and (b) a powdered lipase selected from the group consisting of a powdered lipase which is a lipase derived from Alcaligenes sp., a powdered lipase which is a lipase derived from Rhizopus sp. and a powdered lipase which is a lipase derived from Thermomyces sp. When using this lipase composition, a compound(s) having at least one alcoholic hydroxyl group in the molecule can be effectively esterified with a carboxylic acid(s).
Description
SPECIFICATION
Title of the Invention A lipase powder composition and a process for preparing an esterified compound by using the same Technical Field of the Invention The present invention relates to a powdered lipase composition which can be suitably used in an esterification of a compound(s) having alcoholic hydroxyl group, such as glycerin, with various fatty acids and the like, and to a process for preparing an esterified compound, such as triglyceride, by using the same.
Background of the Invention Lip ases are widely used in the reactions such as esterification of various carboxylic acids such as fatty acids with alcohols such as mono-alcohol and polyalcohol, and trans-esterification between plural carboxylates. Among these, when a lipase powder itself is used in esterification involving dehydrogenation, its activity does not fully express. Further, it is difficult to uniformly disperse the lipase powder into a reaction system, and also to recover it. Therefore, it is common to immobilize a lipase to some carriers, such as anion-exchange resin (Patent Literature 1), phenol adsorption resin (Patent Literature 2), a hydrophobic carrier (Patent Literature 3), cation-exchange resin (Patent Literature 4) and chelate resin (Patent Literature 5) and the like to use it in the reactions such as esterification reaction.
As mentioned above, a lipase has been conventionally immobilized and used in the esterification reaction. However, the immobilized lipase loses an original lipase activity through the immobilization. In addition, when a porous carrier was used, the raw materials and products have gotten stuck in fine pores and, as a result, the ratio of esterification decreases.
In light of the situations mentioned above, various technologies have been developed wherein a lipase powder is used. For example, a trans-esterification method is proposed wherein in the presence or absence of an inactive organic solvent(s), a lipase powder is dispersed into a raw material(s) containing ester in -- the trans-esterification in such a manner that 90% or more of the particles of the dispersed lipase powder can keep particle size of 1 to 100ttm in the reaction (Patent Literature 6). It is also proposed that enzyme powder is used, which is obtained by drying an enzyme solution(s) containing phospholipid and lipid-soluble vitamins (Patent Literature 7).
[Patent Literature 1] Japanese Patent Publication No. Sho 60-98984 [Patent Literature 2] Japanese Patent Publication No. Sho 61-202688 [Patent Literature 3] Japanese Patent Publication No. Hei 2-138986 [Patent Literature 4] Japanese Patent Publication No. Hei 3-61485 [Patent Literature 5] Japanese Patent Publication No. Hei 1-262795 [Patent Literature 6] Japanese Patent No. 2668187 [Patent Literature 7] Japanese Patent Publication No. 2000-106873 Disclosure of the Invention An object of the present invention is to provide a powdered lipase -- composition which allows to effectively conduct an esterification reaction.
Another object of the present invention is to provide a process for preparing an esterified compound by using the above-mentioned lipase composition.
The above objects and other objects will be apparent from the following descriptions.
By using a combination of particular two powdered lipases in place of an immobilized lipase, extremely high ratio of esterification can be accomplished, by which the above-mentioned problems can be solved. The present invention has been completed on the basis of these findings.
Namely, the present invention provides a lipase composition comprising (a) a powdered lipase which is a lipase derived from Rhizomucor sp. or a powdered lipase which is a lipase derived from Penicillium sp. and (b) a powdered lipase selected from the group consisting of a powdered lipase which is a lipase derived from Alcaligenes sp., a powdered lipase which is a lipase derived from Rhizopus sp. and a powdered lipase which is a lipase derived from Thermomyces sp.
The present invention also provides a process for preparing an esterified compound(s), which comprises esterifying a compound(s) having at least one alcoholic hydroxyl group in the molecule, in the presence of the above-mentioned lipase composition, with a carboxylic acid(s).
Brief Description of Drawing Fig. 1 demonstrates production of triglyceride in esterification reaction when the lipase composition comprising a combination of two lipases (RM (1%) + QL
(0.1%) as well as G (0.7%) + QL (0.3%)).
Best Mode for carrying out the Invention As the lipase derived from Rhizomucor sp. which is used as component (a), a lipase derived from Rhizomucor miehei, that is, a 1,3-specific lipase is preferably used. A lipase-containing solution, Palatase (20000L) prepared by Novozymes Japan Co., Ltd. can be made in power form to use the resultant as the powdered lipase. As this powdered lipase, a powder which is spherical and has a water content of not more than 10 % by weight is preferably used. It is more preferable that the water content thereof be 6.5 to 8.5 % by weight.
Heretofore, Rhizomucor miehei sometimes used to belong to Mucor sp.
The above-mentioned powdered lipase can be easily prepared, for example, by spray drying an aqueous solution containing a lipase.
Here, examples of the aqueous solution containing a lipase include a lipase culture solution from which a cell body is removed, a purified culture solution thereof; a solution in which the lipase powder obtained from these culture = CA 02577473 2007-02-14 solutions is dissolved and dispersed again; a solution in which the commercially available lipase powder is dissolved and dispersed again; and a commercially available liquid lipase. In order to enhance lipase activity, it is more preferable that low-molecular-weight components such as salts are removed from the solution. In order to enhance the powder property, it is more preferable that low-molecular-weight components such as sugar are removed from the solution.
A lipase culture solution includes, for example, aqueous solutions containing soybean flour, peptone, corn steep liquor, K2HPO4, (NH4)2SO4, MgSO4 = 71120 and the like. The concentrations thereof are as follows: the soybean flour is 0.1 to 20% by weight and preferably 1.0 to 10% by weight; peptone is 0.1 to 30% by weight and preferably 0.5 to 10% by weight; the corn steep liquor is 0.1 to 30% by weight and preferably 0.5 to 10% by weight; K211PO4 is 0.01 to 20% by weight and preferably 0.1 to 5% by weight; (I\1114)2SO4 is 0.01 to 20% by weight and preferably 0.05 to 5% by weight; and MgSO4 = 71120 is 0.01 to 20% by weight and preferably 0.05 to 5% by weight. The culture conditions thereof should be controlled as follows: the culture temperature is 10 to 40 C and preferably 20 to 35 C; the quantity of airflow is 0.1 to 2.0VVM and preferably 0.1 to 1.5VVM;
the rotation speed for stirring is 100 to 800rpm and preferably 200 to 400rpm; pH
is 3.0 to 10.0 and preferably 4.0 to 9.5.
The separation of a cell body is preferably conducted by centrifugation, the membrane filter procedure and the like.
The removal of the low-molecular-weight components such as salts and sugar can be treated with ultrafiltration membranes. Specifically, after the treatment with ultrafiltration membranes, the aqueous solution containing a lipase is concentrated so as to become 1/2 volume thereof; and then, the same amount of a phosphate buffer as that of the concentrated solution is added thereto. By repeating these procedures once to 5 times, the aqueous solution containing a lipase can be obtained, from which the low-molecular-weight components are removed.
The centrifugation is preferably controlled to 200 to 20,000 x g. The pressure applied to the membrane filter is preferably controlled by microfiltration membranes, the filter press and the like to become not more than 3.0kg/m2. In case of enzymes in the cell body, it is preferable that cell breakage thereof is conducted by the homogenizer, Waring blender, the ultrasonic disruption, the French press, the ball mill and the like; then the cell residues are removed by centrifugation, the membrane filter procedure and the like. The rotation speed of the homogenizer for stirring is 500 to 30,000rpm and preferably 1,000 to 15,000rpm. The rotation speed of Waring blender is 500 to 10,000rpm and preferably 1,000 to 5,000rpm. The time for stirring is 0.5 to 10 minutes and preferably 1 to 5 minutes. It is preferable that the ultrasonic disruption is conducted under the condition of 1 to 50 KHz and more preferably 10 to 20 KHz.
It is preferable that the ball mill has glass pellets having the diameter of 0.1 to 0.5mm.
In the present invention, it is preferable that the aqueous solution containing a lipase is that containing 5 to 30% by weight of lipase as a solid content.
Here, the concentrations of the solid content in the aqueous solution containing a lipase can be determined as Brix.% by using, for example, the sugar content analyzer (Refractometer) (CIS Corporation., Ltd.: BRX-242).
Immediately before the drying process such as spray drying, it is preferable that the aqueous solution containing a lipase is adjusted to pH 6 to 7.5. It is more preferable that it is adjusted to pH 7 or lower and further more preferably to pH 6.5 to 7Ø The pH adjustment can be conducted in any stage before the drying process such as spray drying. The pH of the aqueous solution containing a lipase may be preliminarily adjusted so that pH immediately before the drying process is within the above range. Various alkaline chemicals and acids can be used for the adjustment of pH and alkali metal hydroxides such as sodium hydroxide are preferably used.
In some stage before the drying process, the aqueous solution containing a = 5 =
lipase may be concentrated. The concentration methods are not particularly limited and they include evaporator, flash evaporator, the concentration by ultrafiltration, the concentration by microfiltration, salting out by inorganic salts, precipitation methods with solvents, absorption methods with ion-exchange cellulose and the like, and water absorption methods with water-absorbing gels.
Among these, the concentration by ultrafiltration and evaporator are preferable.
The module for the concentration by ultrafiltration is preferably a flat membrane or a hollow fiber membrane having a fractioned molecular weight of 3,000 to 100,000 and more preferably 6,000 to 50,000. The materials of the membrane are preferably polyacrylonitrile, polysulfonic and the like.
It is preferable that spray drying is conducted by spray dryers such as nozzle countercurrent flow, disk countercurrent flow, nozzle concurrent flow and disk concurrent flow, and the disk concurrent flow is more preferable. The spray drying is preferably controlled as follows: the rotation speed of the atomizer is 4,000 to 20,000rpm; and heating is 100 to 200 C for inlet temperature and 40 to 100 C for outlet temperature.
Another lipase of component (a) used in the present invention is a lipase derived from Penicillium sp. As the lipase, a lipase derived from Penicillium camemberti, that is, a 1,3-specific lipase is preferably used. As this powdered lipase, Lipase G "AMANO" 50 sold by AMANO ENZYME Co., Ltd. and the like can be used. The optical pH of this lipase is 5.0, in particular, the lipase effectively acts between pH 4.5 and 6Ø The optical temperature of the lipase is 40 C. This powdered lipase is a white to fawn-colored fine powder having no carrier.
In addition to this lipase, a lipase derived from Penicillium sp. can be obtained as a powdered material by the method as explained in relation to the above-mentioned lipase derived from Rhizomucor sp.
The lipase of component (b) used in the present invention is a lipase derived from Alcaligenes sp. As a powdered material of this lipase, Lipase QL and Lipase PL of Meito Sangyo Co., Ltd. and the like can be used.
The properties of Lipase QL are as follows: the molecular weight determined by gel filtration is from 180,000 to 190,000, the isoelectric point is 4.1, the optical pH is from 7 to 8.5, the optical temperature is 60 C, the pH stability is from 6 to 10 and the temperature stability is 40 C or less. The properties of Lipase PL
are as follows: the molecular weight determined by gel filtration is from 350,000 to 370,000, the isoelectric point is 4.5, the optical pH is from 7 to 8.5, the optical temperature is from 37 to 40 C, the pH stability is from 7 to 10 and the temperature stability is 40 C or less. Both lipases are a buff yellow fine powder having no carrier. According to the present invention, Lipase QL is preferably used.
In addition to this lipase, a lipase derived from Alcaligenes sp. can be obtained as a powdered material by the method as explained in relation to the above-mentioned lipase derived from Rhizomucor sp.
Another lipase of component (b) used in the present invention a lipase derived from Rhizopus sp. and a lipase derived from Thermomyces sp. As the lipase derived from Rhizopus sp., a lipase derived from Rhizopus oryzae is preferably used. As the powdered material of this lipase, powdered Lipase F-AP15 of AMANO ENZYME Co., Ltd. can be used.
As the lipase derived from Thermomyces sp., a lipase derived from Thermomyces lanuginosus is preferably used. As the powdered material of this lipase, a powdered material obtained by subjecting lipozyme TL (100L) of NOVOZYME Japan Co., Ltd. to membrane treatment and then triturating the resultant by spray dry can be used.
The particle diameter of the powdered lipases of components (a) and (b) is optional. However, 90 % or more of the lipase powders preferably has a particle diameter of 1 to 100 p m. For example, the particle diameter of the powdered lipase can be determined with Particle Size Distribution Analyzer (LA-500) of HORIBA, Ltd.
According to the present invention, as for the proportion of the powdered lipases of components (a) and (b), the weight ratio of component (a) to component (b) is preferably 1:99 to 99:1. In particular, the weight ratio of (a) the powdered lipase which is a lipase derived from Rhizomucor sp. to (b) the powdered lipase which is a lipase derived from Alcaligenes sp. is more preferably 60:40 to 98:2, and most preferably 70:30 to 95:5. In addition, the weight ratio of (a) the powdered lipase which is a lipase derived from Penicillium sp. to (b) the powdered lipase which is a lipase derived from Alcaligenes sp. is more preferably 10:90 to 90:10, and most preferably 70:30 to 30:70. In particular, this combination of these lipases is characterized by extremely wide combination ratio of the two powdered lipases which produces advantageously effects.
The weight ratio of (a) the powdered lipase to (b) the powdered lipase which is a lipase derived from Rhizopus sp. is more preferably 10:90 to 70:30, and most preferably 20:80 to 40:60. In addition, the weight ratio of (a) the powdered lipase to (b) the powdered lipase which is a lipase derived from Thermomyces sp.
is more preferably 10:90 to 90:10, and most preferably 30:70 to 80:20.
The esterification method using the powdered lipase composition of the present invention is explained below.
The compound having at least One alcoholic hydroxyl group in the molecule, which is esterified includes various compounds such as various monoalcohols, polyhydric alcohols, amino alcohols etc. Example of the compound includes short chain, medium chain or long chain, saturated or unsaturated, linear or branched alcohols, polyhydric alcohol such as glycols, glycerin, erythritols.
Among these, glycerin is preferred.
On the other hand, examples of the carboxylic acid include short chain, medium chain or long chain, saturated or unsaturated, linear or branched carboxylic acid. These include C6-30 fatty acid such as octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid etc. These carboxylic acids can be used singly or in combination of two or more. Among these, unsaturated fatty acid is preferred, in particular, use of conjugated linoleic acid is preferred.
The esterification can be conducted, for example, under the condition described in Japanese Patent un-examined Publication (KOKAI) Hei 13-169795 and Japanese Patent un-examined Publication (KOKAI) Hei 15-113396. For one example, based on the total weight of substrate, that is, total weight of the compound(s) having alcoholic hydroxyl group and carboxylic acid(s), 0.1 to 2 %
by weight of the powdered lipase composition according to the present invention is added, and the reaction is conducted at 30 to 60 C for 24 to 72 hours. In this case, the reaction is preferably conducted while water produced by esterification is removed (dewatering) by reducing pressure of the reaction system. Level of depressurization (degree of reduced pressure) is preferably 1 to 100 hPa, more preferably 1 to 50 hPa, and most preferably 1 to 25 hPa. Within this range, dewatering is more preferably conducted while level of depressurization is increased stepwise. Furthermore, dewatering can be more effectively conducted by blowing nitrogen in dewatering treatment. In case where the powdered lipase which is a lipase derived from Penicillium sp. is used, it is preferred that based on the total weight of substrate, 0.1 to 5 % by weight of water is preliminarily added and the reaction is started and then, the reaction is conducted while water produced by esterification is removed.
According to the present invention, extremely high ratio of esterification can be obtained, in particular, triglyceride can be prepared directly from glycerin and carboxylic acid in good yield.
The present invention will be explained in detail by the following Examples.
Preparation example 1 The low-molecular-weight components were removed by using the UF module (Asahi Chemical Industry Co., Ltd.: SIP-0013) from a lipase derived from Rhizomucor miehei of NOVOZYME Japan Co., Ltd., trade name: Palatase (20000L), which was dissolved and dispersed in an aqueous solution to obtain an aqueous solution containing a lipase (the concentration of the solid content:
10.6% by weight). Specifically, liquid lipase (Palatase) was treated with ultrafiltration membranes under cooling with ice and concentrated so as to become 1/2 volume thereof. Then, the same amount of a 0.01M phosphate buffer as that of the concentrated solution was added thereto. The same procedures of ultrafiltration and the addition of a phosphate buffer were conducted three times to the resulting solution to obtain an aqueous solution containing a lipase (the volume ratio: a lipase concentrated solution / buffer = 1/1).
The pH of the aqueous solution containing a lipase was adjusted with an aqueous solution of sodium hydroxide to become the pH 6.8 to 6.9.
Then, the solution was sprayed by using a spray dryer (SD-1000: TOKYO
RIKAKIKAI CO, LTD) under the conditions of inlet temperature: 130 C, the air content for drying: 0.7 to 1.1m3/min, and spray pressure: 11 to 12kpa to obtain lipase powder. The shape of the thus-obtained lipase powder was spherical, 90%
by weight or more of the lipase powder has a particle size of 1 to 100itm and the average particle size thereof was 8.211m. The particle size was determined by Particle Size Distribution Analyzer (LA-500) of HORIBA, Ltd. The water content after the dry heat at 105 C for 1 hour was 7.9% by weight.
The concentration of the solid content in the aqueous solution containing a lipase was determined as Brix.% by using the sugar content analyzer (CIS Co., Ltd.: BRX-242).
Example 1 [Esterification reaction under the nitrogen blowing condition using various lipase or lipase composition]
To reaction vessel with agitator, 10 g of glycerin and 90 g of conjugated linoleic acid were added. To the mixture, the following lipase powder enzyme was added under agitation. Shortly after the resultant was left to stand for minutes while the temperature was kept at about 45 C in water bath, depressurization was conducted by depressurizing pump. At that time, nitrogen was blown to allow easy production of water.
At first, level of depressurization was kept at about 40 hPa. After 2 to 3 hours, the temperature was gradually increased to about 50 C. After additional 2 to 3 hours, the temperature was increased to about 60 C. Then, this temperature was kept. On the other hand, level of depressurization was gradually increased, finally to about 10 hPa.
If G, or G+QL was used as enzyme, before reaction, water of 2 % by weight was added thereto based on total weight of glycerin and conjugated linoleic acid.
The progress of esterification reaction was confirmed by GLC analysis while sampling was conducted as needed. In this case, percentages of unreacted glycerin, MG, DG, TG were determined, and production rate of TG was shown in the following Table-1 and Fig.l.
Used lipase RM (1%): The powdered lipase derived from Rhizomucor miehei which belongs to Rhizomucor sp., prepared by Preparation Example 1 was singly used.
Numeric values shown in parenthesis indicate amount of used lipase. If said numeric value is 1 %, it is meant that the amount of use lipase is 1 % by weight based on the total weight of glycerin and conjugated linoleic acid (hereinafter, the same).
RM (1%) + QL (0.1%) (present invention): Combination of the powdered lipase derived from Rhizomucor miehei which belongs to Rhizomucor sp., prepared by Preparation Example 1 and the powdered lipase QL derived from Alcaligenes sp., prepared by Meito Sangyo Co., Ltd. was used. The used amount was shown in parenthesis (hereinafter, the same).
G (1%): The powdered lipase G "AMANO" 50 derived from Penicillium sp., prepared by AMANO ENZYME Co., Ltd. was singly used.
a (0.7%) + QL (0.3%) (present invention): Combination of the powdered lipase G "AMANO" 50 derived from Penicillium sp., prepared by AMANO
ENZYME Co., Ltd. and the powdered lipase QL derived from Alcaligenes sp., prepared by Meito Sangyo Co., Ltd. was used.
QL (1%): The powdered lipase QL derived from Alcaligenes sp., prepared by Meito Sangyo Co., Ltd. was singly used.
Table-1 Reaction RM (1%) RM (1%) + G(1%) G (0.7%) + QL (1%) time (h) QL (0.1%) QL (0.3%) 5.5 25 From the result shown in Table 1, it is found that yield of triglyceride after 44 hours was greatly increased, in particular, from 75 % to 91 % when a combination of the powdered lipase derived from Rhizomucor miehei which belongs to Rhizomucor sp. and the powdered lipase QL derived from Alcaligenes sp. (RM (1%) + QL (0.1%)) was used in place of singly used powdered lipase derived from Rhizomucor miehei which belongs to Rhizomucor sp. (RM (1%)).
Furthermore, in light of triglyceride production, the time that the triglyceride production reached 75 % was over 40 hours in case of RM (1%). On the other hand, in case of RM (1%) + QL (0.1%), the time was half of that of RM (1%), that is, 20 hours.
For those skilled in the art, it was surprising that even if the powdered lipase QL derived from Alcaligenes sp. was singly used in an amount of 1%, triglyceride was hardly produced.
On the other hand, for those skilled in the art, it was surprising that even if the powdered lipase G "AMANO" 50 derived from Penicillium sp. (G (1%)) or the powdered lipase QL derived from Alcaligenes sp. (QL (1%)) was singly used, triglyceride was hardly produced, but if a combination of these lipases (G
(0.7%) +
QL (0.3%)) was used, the yield of triglyceride after 44 hours was very high, in particular, 87 %.
Example 2 [Esterification reaction using RM or various lipase composition]
To conduct esterification reaction, the same procedure was conducted as that of Example 1 except that nitrogen gas blowing was not conducted, water was added or not added, and final depressurization levels were those shown in Table-2. Sampling was conducted after 47 hours from starting the reaction, and the production rate of TG was confirmed by GLC analysis. In this case, percentages of unreacted glycerin, MG, DG, TG were determined. The production rate of TG was shown in the following Table-2. In Table-2, in case where the description "water added" does not appear, water did not added. In addition, the numeric values expressed as % in item of Enzyme in Table-2 indicate used amount of lipase. If the numeric value is 1 %, used amount of lipase is 1 % by weight based on the total weight of glycerin and conjugated linoleic acid. Furthermore, the numeric ratio described in parenthesis in item of Enzyme in Table-2 indicates the weight ratio of various lipase combinations.
Used lipase RM: The powdered lipase derived from Rhizomucor miehei which belongs to Rhizomucor sp., which is the same as that of Example 1.
QL: The powdered lipase QL derived from Alcaligenes sp., prepared by Meito Sangyo Co., Ltd., which is the same as that of Example 1.
G: The powdered lipase G "AMANO" 50 derived from Penicillium sp., prepared by AMANO ENZYME Co., Ltd., which is the same as that of Example 1.
PL: The powdered lipase QL derived from Alcaligenes sp., prepared by Meito Sangyo Co., Ltd.
TL: The powder obtained by subjecting lipozyme TL (100L) derived from Thermomyces lanuginosus, prepared by subjecting NOVOZYME Japan Co., Ltd.
to membrane treatment and then spray dry.
F-AP: The powdered lipase F-AP15 derived from Rhizopus oryzae, prepared by AMANO ENZYME Co., Ltd.
Table-2 Enzyme TG% Depressurization hPa RM singly used 1% 69 20 RM+QL (1%, 8:2) 82 19 G+QL (1%, 5:5) water 80 19 added 2%
RM+PL (1%, 8:2) 83 17 G+TL (1%, 5:5) water 89 5 added 1%
G+F-AP (1%, 3:7) water 83 3 added 2%
RM+TL (0.6%, 8:2) 72 16 From the result shown in Table-2, it is clear that a combination of the particular lipases (a) and (b) improves esterification ratio.
Title of the Invention A lipase powder composition and a process for preparing an esterified compound by using the same Technical Field of the Invention The present invention relates to a powdered lipase composition which can be suitably used in an esterification of a compound(s) having alcoholic hydroxyl group, such as glycerin, with various fatty acids and the like, and to a process for preparing an esterified compound, such as triglyceride, by using the same.
Background of the Invention Lip ases are widely used in the reactions such as esterification of various carboxylic acids such as fatty acids with alcohols such as mono-alcohol and polyalcohol, and trans-esterification between plural carboxylates. Among these, when a lipase powder itself is used in esterification involving dehydrogenation, its activity does not fully express. Further, it is difficult to uniformly disperse the lipase powder into a reaction system, and also to recover it. Therefore, it is common to immobilize a lipase to some carriers, such as anion-exchange resin (Patent Literature 1), phenol adsorption resin (Patent Literature 2), a hydrophobic carrier (Patent Literature 3), cation-exchange resin (Patent Literature 4) and chelate resin (Patent Literature 5) and the like to use it in the reactions such as esterification reaction.
As mentioned above, a lipase has been conventionally immobilized and used in the esterification reaction. However, the immobilized lipase loses an original lipase activity through the immobilization. In addition, when a porous carrier was used, the raw materials and products have gotten stuck in fine pores and, as a result, the ratio of esterification decreases.
In light of the situations mentioned above, various technologies have been developed wherein a lipase powder is used. For example, a trans-esterification method is proposed wherein in the presence or absence of an inactive organic solvent(s), a lipase powder is dispersed into a raw material(s) containing ester in -- the trans-esterification in such a manner that 90% or more of the particles of the dispersed lipase powder can keep particle size of 1 to 100ttm in the reaction (Patent Literature 6). It is also proposed that enzyme powder is used, which is obtained by drying an enzyme solution(s) containing phospholipid and lipid-soluble vitamins (Patent Literature 7).
[Patent Literature 1] Japanese Patent Publication No. Sho 60-98984 [Patent Literature 2] Japanese Patent Publication No. Sho 61-202688 [Patent Literature 3] Japanese Patent Publication No. Hei 2-138986 [Patent Literature 4] Japanese Patent Publication No. Hei 3-61485 [Patent Literature 5] Japanese Patent Publication No. Hei 1-262795 [Patent Literature 6] Japanese Patent No. 2668187 [Patent Literature 7] Japanese Patent Publication No. 2000-106873 Disclosure of the Invention An object of the present invention is to provide a powdered lipase -- composition which allows to effectively conduct an esterification reaction.
Another object of the present invention is to provide a process for preparing an esterified compound by using the above-mentioned lipase composition.
The above objects and other objects will be apparent from the following descriptions.
By using a combination of particular two powdered lipases in place of an immobilized lipase, extremely high ratio of esterification can be accomplished, by which the above-mentioned problems can be solved. The present invention has been completed on the basis of these findings.
Namely, the present invention provides a lipase composition comprising (a) a powdered lipase which is a lipase derived from Rhizomucor sp. or a powdered lipase which is a lipase derived from Penicillium sp. and (b) a powdered lipase selected from the group consisting of a powdered lipase which is a lipase derived from Alcaligenes sp., a powdered lipase which is a lipase derived from Rhizopus sp. and a powdered lipase which is a lipase derived from Thermomyces sp.
The present invention also provides a process for preparing an esterified compound(s), which comprises esterifying a compound(s) having at least one alcoholic hydroxyl group in the molecule, in the presence of the above-mentioned lipase composition, with a carboxylic acid(s).
Brief Description of Drawing Fig. 1 demonstrates production of triglyceride in esterification reaction when the lipase composition comprising a combination of two lipases (RM (1%) + QL
(0.1%) as well as G (0.7%) + QL (0.3%)).
Best Mode for carrying out the Invention As the lipase derived from Rhizomucor sp. which is used as component (a), a lipase derived from Rhizomucor miehei, that is, a 1,3-specific lipase is preferably used. A lipase-containing solution, Palatase (20000L) prepared by Novozymes Japan Co., Ltd. can be made in power form to use the resultant as the powdered lipase. As this powdered lipase, a powder which is spherical and has a water content of not more than 10 % by weight is preferably used. It is more preferable that the water content thereof be 6.5 to 8.5 % by weight.
Heretofore, Rhizomucor miehei sometimes used to belong to Mucor sp.
The above-mentioned powdered lipase can be easily prepared, for example, by spray drying an aqueous solution containing a lipase.
Here, examples of the aqueous solution containing a lipase include a lipase culture solution from which a cell body is removed, a purified culture solution thereof; a solution in which the lipase powder obtained from these culture = CA 02577473 2007-02-14 solutions is dissolved and dispersed again; a solution in which the commercially available lipase powder is dissolved and dispersed again; and a commercially available liquid lipase. In order to enhance lipase activity, it is more preferable that low-molecular-weight components such as salts are removed from the solution. In order to enhance the powder property, it is more preferable that low-molecular-weight components such as sugar are removed from the solution.
A lipase culture solution includes, for example, aqueous solutions containing soybean flour, peptone, corn steep liquor, K2HPO4, (NH4)2SO4, MgSO4 = 71120 and the like. The concentrations thereof are as follows: the soybean flour is 0.1 to 20% by weight and preferably 1.0 to 10% by weight; peptone is 0.1 to 30% by weight and preferably 0.5 to 10% by weight; the corn steep liquor is 0.1 to 30% by weight and preferably 0.5 to 10% by weight; K211PO4 is 0.01 to 20% by weight and preferably 0.1 to 5% by weight; (I\1114)2SO4 is 0.01 to 20% by weight and preferably 0.05 to 5% by weight; and MgSO4 = 71120 is 0.01 to 20% by weight and preferably 0.05 to 5% by weight. The culture conditions thereof should be controlled as follows: the culture temperature is 10 to 40 C and preferably 20 to 35 C; the quantity of airflow is 0.1 to 2.0VVM and preferably 0.1 to 1.5VVM;
the rotation speed for stirring is 100 to 800rpm and preferably 200 to 400rpm; pH
is 3.0 to 10.0 and preferably 4.0 to 9.5.
The separation of a cell body is preferably conducted by centrifugation, the membrane filter procedure and the like.
The removal of the low-molecular-weight components such as salts and sugar can be treated with ultrafiltration membranes. Specifically, after the treatment with ultrafiltration membranes, the aqueous solution containing a lipase is concentrated so as to become 1/2 volume thereof; and then, the same amount of a phosphate buffer as that of the concentrated solution is added thereto. By repeating these procedures once to 5 times, the aqueous solution containing a lipase can be obtained, from which the low-molecular-weight components are removed.
The centrifugation is preferably controlled to 200 to 20,000 x g. The pressure applied to the membrane filter is preferably controlled by microfiltration membranes, the filter press and the like to become not more than 3.0kg/m2. In case of enzymes in the cell body, it is preferable that cell breakage thereof is conducted by the homogenizer, Waring blender, the ultrasonic disruption, the French press, the ball mill and the like; then the cell residues are removed by centrifugation, the membrane filter procedure and the like. The rotation speed of the homogenizer for stirring is 500 to 30,000rpm and preferably 1,000 to 15,000rpm. The rotation speed of Waring blender is 500 to 10,000rpm and preferably 1,000 to 5,000rpm. The time for stirring is 0.5 to 10 minutes and preferably 1 to 5 minutes. It is preferable that the ultrasonic disruption is conducted under the condition of 1 to 50 KHz and more preferably 10 to 20 KHz.
It is preferable that the ball mill has glass pellets having the diameter of 0.1 to 0.5mm.
In the present invention, it is preferable that the aqueous solution containing a lipase is that containing 5 to 30% by weight of lipase as a solid content.
Here, the concentrations of the solid content in the aqueous solution containing a lipase can be determined as Brix.% by using, for example, the sugar content analyzer (Refractometer) (CIS Corporation., Ltd.: BRX-242).
Immediately before the drying process such as spray drying, it is preferable that the aqueous solution containing a lipase is adjusted to pH 6 to 7.5. It is more preferable that it is adjusted to pH 7 or lower and further more preferably to pH 6.5 to 7Ø The pH adjustment can be conducted in any stage before the drying process such as spray drying. The pH of the aqueous solution containing a lipase may be preliminarily adjusted so that pH immediately before the drying process is within the above range. Various alkaline chemicals and acids can be used for the adjustment of pH and alkali metal hydroxides such as sodium hydroxide are preferably used.
In some stage before the drying process, the aqueous solution containing a = 5 =
lipase may be concentrated. The concentration methods are not particularly limited and they include evaporator, flash evaporator, the concentration by ultrafiltration, the concentration by microfiltration, salting out by inorganic salts, precipitation methods with solvents, absorption methods with ion-exchange cellulose and the like, and water absorption methods with water-absorbing gels.
Among these, the concentration by ultrafiltration and evaporator are preferable.
The module for the concentration by ultrafiltration is preferably a flat membrane or a hollow fiber membrane having a fractioned molecular weight of 3,000 to 100,000 and more preferably 6,000 to 50,000. The materials of the membrane are preferably polyacrylonitrile, polysulfonic and the like.
It is preferable that spray drying is conducted by spray dryers such as nozzle countercurrent flow, disk countercurrent flow, nozzle concurrent flow and disk concurrent flow, and the disk concurrent flow is more preferable. The spray drying is preferably controlled as follows: the rotation speed of the atomizer is 4,000 to 20,000rpm; and heating is 100 to 200 C for inlet temperature and 40 to 100 C for outlet temperature.
Another lipase of component (a) used in the present invention is a lipase derived from Penicillium sp. As the lipase, a lipase derived from Penicillium camemberti, that is, a 1,3-specific lipase is preferably used. As this powdered lipase, Lipase G "AMANO" 50 sold by AMANO ENZYME Co., Ltd. and the like can be used. The optical pH of this lipase is 5.0, in particular, the lipase effectively acts between pH 4.5 and 6Ø The optical temperature of the lipase is 40 C. This powdered lipase is a white to fawn-colored fine powder having no carrier.
In addition to this lipase, a lipase derived from Penicillium sp. can be obtained as a powdered material by the method as explained in relation to the above-mentioned lipase derived from Rhizomucor sp.
The lipase of component (b) used in the present invention is a lipase derived from Alcaligenes sp. As a powdered material of this lipase, Lipase QL and Lipase PL of Meito Sangyo Co., Ltd. and the like can be used.
The properties of Lipase QL are as follows: the molecular weight determined by gel filtration is from 180,000 to 190,000, the isoelectric point is 4.1, the optical pH is from 7 to 8.5, the optical temperature is 60 C, the pH stability is from 6 to 10 and the temperature stability is 40 C or less. The properties of Lipase PL
are as follows: the molecular weight determined by gel filtration is from 350,000 to 370,000, the isoelectric point is 4.5, the optical pH is from 7 to 8.5, the optical temperature is from 37 to 40 C, the pH stability is from 7 to 10 and the temperature stability is 40 C or less. Both lipases are a buff yellow fine powder having no carrier. According to the present invention, Lipase QL is preferably used.
In addition to this lipase, a lipase derived from Alcaligenes sp. can be obtained as a powdered material by the method as explained in relation to the above-mentioned lipase derived from Rhizomucor sp.
Another lipase of component (b) used in the present invention a lipase derived from Rhizopus sp. and a lipase derived from Thermomyces sp. As the lipase derived from Rhizopus sp., a lipase derived from Rhizopus oryzae is preferably used. As the powdered material of this lipase, powdered Lipase F-AP15 of AMANO ENZYME Co., Ltd. can be used.
As the lipase derived from Thermomyces sp., a lipase derived from Thermomyces lanuginosus is preferably used. As the powdered material of this lipase, a powdered material obtained by subjecting lipozyme TL (100L) of NOVOZYME Japan Co., Ltd. to membrane treatment and then triturating the resultant by spray dry can be used.
The particle diameter of the powdered lipases of components (a) and (b) is optional. However, 90 % or more of the lipase powders preferably has a particle diameter of 1 to 100 p m. For example, the particle diameter of the powdered lipase can be determined with Particle Size Distribution Analyzer (LA-500) of HORIBA, Ltd.
According to the present invention, as for the proportion of the powdered lipases of components (a) and (b), the weight ratio of component (a) to component (b) is preferably 1:99 to 99:1. In particular, the weight ratio of (a) the powdered lipase which is a lipase derived from Rhizomucor sp. to (b) the powdered lipase which is a lipase derived from Alcaligenes sp. is more preferably 60:40 to 98:2, and most preferably 70:30 to 95:5. In addition, the weight ratio of (a) the powdered lipase which is a lipase derived from Penicillium sp. to (b) the powdered lipase which is a lipase derived from Alcaligenes sp. is more preferably 10:90 to 90:10, and most preferably 70:30 to 30:70. In particular, this combination of these lipases is characterized by extremely wide combination ratio of the two powdered lipases which produces advantageously effects.
The weight ratio of (a) the powdered lipase to (b) the powdered lipase which is a lipase derived from Rhizopus sp. is more preferably 10:90 to 70:30, and most preferably 20:80 to 40:60. In addition, the weight ratio of (a) the powdered lipase to (b) the powdered lipase which is a lipase derived from Thermomyces sp.
is more preferably 10:90 to 90:10, and most preferably 30:70 to 80:20.
The esterification method using the powdered lipase composition of the present invention is explained below.
The compound having at least One alcoholic hydroxyl group in the molecule, which is esterified includes various compounds such as various monoalcohols, polyhydric alcohols, amino alcohols etc. Example of the compound includes short chain, medium chain or long chain, saturated or unsaturated, linear or branched alcohols, polyhydric alcohol such as glycols, glycerin, erythritols.
Among these, glycerin is preferred.
On the other hand, examples of the carboxylic acid include short chain, medium chain or long chain, saturated or unsaturated, linear or branched carboxylic acid. These include C6-30 fatty acid such as octanoic acid, decanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid etc. These carboxylic acids can be used singly or in combination of two or more. Among these, unsaturated fatty acid is preferred, in particular, use of conjugated linoleic acid is preferred.
The esterification can be conducted, for example, under the condition described in Japanese Patent un-examined Publication (KOKAI) Hei 13-169795 and Japanese Patent un-examined Publication (KOKAI) Hei 15-113396. For one example, based on the total weight of substrate, that is, total weight of the compound(s) having alcoholic hydroxyl group and carboxylic acid(s), 0.1 to 2 %
by weight of the powdered lipase composition according to the present invention is added, and the reaction is conducted at 30 to 60 C for 24 to 72 hours. In this case, the reaction is preferably conducted while water produced by esterification is removed (dewatering) by reducing pressure of the reaction system. Level of depressurization (degree of reduced pressure) is preferably 1 to 100 hPa, more preferably 1 to 50 hPa, and most preferably 1 to 25 hPa. Within this range, dewatering is more preferably conducted while level of depressurization is increased stepwise. Furthermore, dewatering can be more effectively conducted by blowing nitrogen in dewatering treatment. In case where the powdered lipase which is a lipase derived from Penicillium sp. is used, it is preferred that based on the total weight of substrate, 0.1 to 5 % by weight of water is preliminarily added and the reaction is started and then, the reaction is conducted while water produced by esterification is removed.
According to the present invention, extremely high ratio of esterification can be obtained, in particular, triglyceride can be prepared directly from glycerin and carboxylic acid in good yield.
The present invention will be explained in detail by the following Examples.
Preparation example 1 The low-molecular-weight components were removed by using the UF module (Asahi Chemical Industry Co., Ltd.: SIP-0013) from a lipase derived from Rhizomucor miehei of NOVOZYME Japan Co., Ltd., trade name: Palatase (20000L), which was dissolved and dispersed in an aqueous solution to obtain an aqueous solution containing a lipase (the concentration of the solid content:
10.6% by weight). Specifically, liquid lipase (Palatase) was treated with ultrafiltration membranes under cooling with ice and concentrated so as to become 1/2 volume thereof. Then, the same amount of a 0.01M phosphate buffer as that of the concentrated solution was added thereto. The same procedures of ultrafiltration and the addition of a phosphate buffer were conducted three times to the resulting solution to obtain an aqueous solution containing a lipase (the volume ratio: a lipase concentrated solution / buffer = 1/1).
The pH of the aqueous solution containing a lipase was adjusted with an aqueous solution of sodium hydroxide to become the pH 6.8 to 6.9.
Then, the solution was sprayed by using a spray dryer (SD-1000: TOKYO
RIKAKIKAI CO, LTD) under the conditions of inlet temperature: 130 C, the air content for drying: 0.7 to 1.1m3/min, and spray pressure: 11 to 12kpa to obtain lipase powder. The shape of the thus-obtained lipase powder was spherical, 90%
by weight or more of the lipase powder has a particle size of 1 to 100itm and the average particle size thereof was 8.211m. The particle size was determined by Particle Size Distribution Analyzer (LA-500) of HORIBA, Ltd. The water content after the dry heat at 105 C for 1 hour was 7.9% by weight.
The concentration of the solid content in the aqueous solution containing a lipase was determined as Brix.% by using the sugar content analyzer (CIS Co., Ltd.: BRX-242).
Example 1 [Esterification reaction under the nitrogen blowing condition using various lipase or lipase composition]
To reaction vessel with agitator, 10 g of glycerin and 90 g of conjugated linoleic acid were added. To the mixture, the following lipase powder enzyme was added under agitation. Shortly after the resultant was left to stand for minutes while the temperature was kept at about 45 C in water bath, depressurization was conducted by depressurizing pump. At that time, nitrogen was blown to allow easy production of water.
At first, level of depressurization was kept at about 40 hPa. After 2 to 3 hours, the temperature was gradually increased to about 50 C. After additional 2 to 3 hours, the temperature was increased to about 60 C. Then, this temperature was kept. On the other hand, level of depressurization was gradually increased, finally to about 10 hPa.
If G, or G+QL was used as enzyme, before reaction, water of 2 % by weight was added thereto based on total weight of glycerin and conjugated linoleic acid.
The progress of esterification reaction was confirmed by GLC analysis while sampling was conducted as needed. In this case, percentages of unreacted glycerin, MG, DG, TG were determined, and production rate of TG was shown in the following Table-1 and Fig.l.
Used lipase RM (1%): The powdered lipase derived from Rhizomucor miehei which belongs to Rhizomucor sp., prepared by Preparation Example 1 was singly used.
Numeric values shown in parenthesis indicate amount of used lipase. If said numeric value is 1 %, it is meant that the amount of use lipase is 1 % by weight based on the total weight of glycerin and conjugated linoleic acid (hereinafter, the same).
RM (1%) + QL (0.1%) (present invention): Combination of the powdered lipase derived from Rhizomucor miehei which belongs to Rhizomucor sp., prepared by Preparation Example 1 and the powdered lipase QL derived from Alcaligenes sp., prepared by Meito Sangyo Co., Ltd. was used. The used amount was shown in parenthesis (hereinafter, the same).
G (1%): The powdered lipase G "AMANO" 50 derived from Penicillium sp., prepared by AMANO ENZYME Co., Ltd. was singly used.
a (0.7%) + QL (0.3%) (present invention): Combination of the powdered lipase G "AMANO" 50 derived from Penicillium sp., prepared by AMANO
ENZYME Co., Ltd. and the powdered lipase QL derived from Alcaligenes sp., prepared by Meito Sangyo Co., Ltd. was used.
QL (1%): The powdered lipase QL derived from Alcaligenes sp., prepared by Meito Sangyo Co., Ltd. was singly used.
Table-1 Reaction RM (1%) RM (1%) + G(1%) G (0.7%) + QL (1%) time (h) QL (0.1%) QL (0.3%) 5.5 25 From the result shown in Table 1, it is found that yield of triglyceride after 44 hours was greatly increased, in particular, from 75 % to 91 % when a combination of the powdered lipase derived from Rhizomucor miehei which belongs to Rhizomucor sp. and the powdered lipase QL derived from Alcaligenes sp. (RM (1%) + QL (0.1%)) was used in place of singly used powdered lipase derived from Rhizomucor miehei which belongs to Rhizomucor sp. (RM (1%)).
Furthermore, in light of triglyceride production, the time that the triglyceride production reached 75 % was over 40 hours in case of RM (1%). On the other hand, in case of RM (1%) + QL (0.1%), the time was half of that of RM (1%), that is, 20 hours.
For those skilled in the art, it was surprising that even if the powdered lipase QL derived from Alcaligenes sp. was singly used in an amount of 1%, triglyceride was hardly produced.
On the other hand, for those skilled in the art, it was surprising that even if the powdered lipase G "AMANO" 50 derived from Penicillium sp. (G (1%)) or the powdered lipase QL derived from Alcaligenes sp. (QL (1%)) was singly used, triglyceride was hardly produced, but if a combination of these lipases (G
(0.7%) +
QL (0.3%)) was used, the yield of triglyceride after 44 hours was very high, in particular, 87 %.
Example 2 [Esterification reaction using RM or various lipase composition]
To conduct esterification reaction, the same procedure was conducted as that of Example 1 except that nitrogen gas blowing was not conducted, water was added or not added, and final depressurization levels were those shown in Table-2. Sampling was conducted after 47 hours from starting the reaction, and the production rate of TG was confirmed by GLC analysis. In this case, percentages of unreacted glycerin, MG, DG, TG were determined. The production rate of TG was shown in the following Table-2. In Table-2, in case where the description "water added" does not appear, water did not added. In addition, the numeric values expressed as % in item of Enzyme in Table-2 indicate used amount of lipase. If the numeric value is 1 %, used amount of lipase is 1 % by weight based on the total weight of glycerin and conjugated linoleic acid. Furthermore, the numeric ratio described in parenthesis in item of Enzyme in Table-2 indicates the weight ratio of various lipase combinations.
Used lipase RM: The powdered lipase derived from Rhizomucor miehei which belongs to Rhizomucor sp., which is the same as that of Example 1.
QL: The powdered lipase QL derived from Alcaligenes sp., prepared by Meito Sangyo Co., Ltd., which is the same as that of Example 1.
G: The powdered lipase G "AMANO" 50 derived from Penicillium sp., prepared by AMANO ENZYME Co., Ltd., which is the same as that of Example 1.
PL: The powdered lipase QL derived from Alcaligenes sp., prepared by Meito Sangyo Co., Ltd.
TL: The powder obtained by subjecting lipozyme TL (100L) derived from Thermomyces lanuginosus, prepared by subjecting NOVOZYME Japan Co., Ltd.
to membrane treatment and then spray dry.
F-AP: The powdered lipase F-AP15 derived from Rhizopus oryzae, prepared by AMANO ENZYME Co., Ltd.
Table-2 Enzyme TG% Depressurization hPa RM singly used 1% 69 20 RM+QL (1%, 8:2) 82 19 G+QL (1%, 5:5) water 80 19 added 2%
RM+PL (1%, 8:2) 83 17 G+TL (1%, 5:5) water 89 5 added 1%
G+F-AP (1%, 3:7) water 83 3 added 2%
RM+TL (0.6%, 8:2) 72 16 From the result shown in Table-2, it is clear that a combination of the particular lipases (a) and (b) improves esterification ratio.
Claims (12)
1. A lipase composition comprising (a) a powdered lipase which is a lipase from Rhizomucor sp. or a powdered lipase which is a lipase from Penicillium sp. and (b) a powdered lipase selected from the group consisting of a powdered lipase which is a lipase from Alcaligenes sp., a powdered lipase which is a lipase from Rhizopus sp. and a powdered lipase which is a lipase from Thermomyces sp.
2. The lipase composition according to claim 1, wherein the powdered lipase (b) is a powdered lipase from Alcaligenes sp.
3. The lipase composition according to claim 1 or 2, wherein the powdered lipase (a) is a lipase from Rhizomucor miehei.
4. The lipase composition according to claim 1 or 2, wherein the lipase from Penicillium sp. is a lipase from Penicillium camemberti.
5. The lipase composition according to any one of claims 1 to 4, wherein the powdered lipases of (a) and (b) are not supported on carriers.
6. The lipase composition according to any one of claims 1 to 5, wherein the water contents of the powdered lipases of (a) and (b) are 10 % by weight or less.
7. The lipase composition according to any one of claims 1 to 6, wherein 90 %
by weight or more of the powdered lipases have a particle size of 1 to 100 m.
by weight or more of the powdered lipases have a particle size of 1 to 100 m.
8. The lipase composition according to any one of claims 1 to 7, which is used for esterification.
9. A process for preparing an esterified compound(s), which comprises esterifying a compound(s) having at least one alcoholic hydroxyl group, in the presence of the lipase composition according to any one of claims 1 to 7, with a carboxylic acid(s).
10. The process according to claim 9, wherein the compound having at least one alcoholic hydroxyl group is glycerin.
11. The process according to claim 9 or 10, wherein the carboxylic acid is unsaturated fatty acid.
12. The process according to any one of claims 9 to 11, wherein the esterification is conducted under reduced pressure, wherein level of depressurization is 1 to 100 hPa.
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JP2004-243895 | 2004-08-24 | ||
JP2004243895 | 2004-08-24 | ||
JP2005-091881 | 2005-03-28 | ||
JP2005091881A JP4394598B2 (en) | 2004-08-24 | 2005-03-28 | Lipase powder composition and method for producing esterified product using the same |
PCT/JP2005/014936 WO2006022166A1 (en) | 2004-08-24 | 2005-08-16 | Powdery lipase composition and method of producing esterified product by using the same |
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EP (1) | EP1790718B1 (en) |
JP (1) | JP4394598B2 (en) |
KR (1) | KR101174532B1 (en) |
CN (1) | CN101006173B (en) |
AT (1) | ATE428772T1 (en) |
AU (1) | AU2005275919A1 (en) |
CA (1) | CA2577473C (en) |
DE (1) | DE602005013981D1 (en) |
DK (1) | DK1790718T3 (en) |
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TWI438279B (en) * | 2007-03-16 | 2014-05-21 | Nisshin Oillio Group Ltd | Lipase powder preparation, method of preparin the same and use thereof |
JP2009065887A (en) * | 2007-09-12 | 2009-04-02 | Bio−energy株式会社 | Method for producing fatty acid ester |
TWI403578B (en) * | 2009-08-05 | 2013-08-01 | Standard Foods Corp | Method of producing diglycerides from the by-product of edible oil refining |
US9416337B2 (en) * | 2012-10-10 | 2016-08-16 | Basf Se | Ester synthesis |
GB201501081D0 (en) * | 2015-01-22 | 2015-03-11 | Cilian Ag | Use of enzymes with a wide pH activity range as medicaments for promoting digestion |
CN106566653A (en) * | 2016-11-03 | 2017-04-19 | 山东龙大粮油有限公司 | Oil dewatering and drying method capable of reducing loss of volatile fragrant components in oil |
CN108239663B (en) * | 2016-12-23 | 2022-07-08 | 丰益(上海)生物技术研发中心有限公司 | Method for hydrolyzing high-melting-point grease by enzyme method |
CN108239578B (en) * | 2016-12-23 | 2022-06-07 | 丰益(上海)生物技术研发中心有限公司 | Method for improving hydrolysis rate of grease |
KR20230085877A (en) * | 2021-12-07 | 2023-06-14 | 주식회사 종근당 | Method for producing high-purity triglyceride derivatives containing high content of eicosapentaenoic acid (EPA) |
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FR2449726A1 (en) * | 1979-02-22 | 1980-09-19 | Millipore Corp | Fast reacting lipase compsn. for hydrolysis of glycerol ester(s) - contg. Rhizopus arrhizus lipase and Pseudomonas Fluoresens lipase |
US4394445A (en) * | 1979-02-22 | 1983-07-19 | Nix Paul T | Enzymatic glyceride hydrolysis |
DK402583D0 (en) | 1983-09-05 | 1983-09-05 | Novo Industri As | PROCEDURE FOR THE MANUFACTURING OF AN IMMOBILIZED LIPASE PREPARATION AND APPLICATION |
DK153762C (en) | 1985-02-27 | 1989-01-09 | Novo Industri As | PROCEDURE FOR PREPARING AN IMMOBILIZED LIPASE PREPARATION |
GB8729890D0 (en) | 1987-12-22 | 1988-02-03 | Unilever Plc | Improvements in & relating to fat processes |
JP2749587B2 (en) | 1988-04-11 | 1998-05-13 | 花王株式会社 | Method for producing immobilized enzyme |
JP2794201B2 (en) | 1989-07-31 | 1998-09-03 | 味の素株式会社 | Immobilized lipase enzyme preparation |
JP2668187B2 (en) * | 1993-09-17 | 1997-10-27 | 日清製油株式会社 | Transesterification method using lipase powder |
US6495357B1 (en) * | 1995-07-14 | 2002-12-17 | Novozyme A/S | Lipolytic enzymes |
JP2000106873A (en) | 1998-10-06 | 2000-04-18 | Nisshin Oil Mills Ltd:The | Thermally stable enzyme and production thereof |
WO2002000908A2 (en) * | 2000-09-25 | 2002-01-03 | Novozymes A/S | Methods for processing crustacean material |
CN1774508A (en) * | 2002-08-16 | 2006-05-17 | 诺维信北美公司 | Process for enzymatic hydrolysis of cyclic oligomers |
JP2004283043A (en) * | 2003-03-20 | 2004-10-14 | Nof Corp | Method for producing medium chain fatty acid-bound phospholipid |
CN1239710C (en) * | 2003-11-25 | 2006-02-01 | 东莞新宝精化有限公司 | Diglyceride lipin production method |
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JP4394598B2 (en) | 2010-01-06 |
ATE428772T1 (en) | 2009-05-15 |
TW200619387A (en) | 2006-06-16 |
TWI347974B (en) | 2011-09-01 |
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JP2006087423A (en) | 2006-04-06 |
CA2577473A1 (en) | 2006-03-02 |
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DE602005013981D1 (en) | 2009-05-28 |
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